Biomedical subjects
Publications and source records attributed to W M Williams.
A case of germ cell neoplasm in an undescended retroperitoneal testicle is reported. CT revealed a large mass most consistent with a chronic hematoma. MRI demonstrated findings typical for neoplasm, and this was confirmed on biopsy.
In our efforts to identify products that might be used for active immunotherapy in human immunodeficiency virus (HIV) infection, we have studied synthetic peptides derived from the CD4 attachment site of gp120. Two peptides have emerged with particularly interesting properties. The first (B138) is linear and spans the envelope residues 421-438; the second (1005/45) encompasses amino acids 418-445 and is cyclized by way of a disulphide bond joining its terminal cysteines. Both species have been shown to inhibit syncytial formation in a conventional bioassay, B138 being the most efficient. Both peptides elicit high titres of anti-peptide antibodies in immunized mice, rabbits and goats, with titres exceeding 1:10(5) in many cases. A substantial portion of this response is directed against gp120 as determined by enzyme-linked immunosorbent assay (ELISA). Analysis by flow cytometry has demonstrated that the antisera are broadly reactive with multiple diverse strains of HIV. The anti-gp120 activity of the anti-peptide antiserum was further confirmed by radioimmuno-precipitation (RIP) assays. Furthermore, RIP analysis and inhibition experiments in a GD4-gp120 binding assay have revealed that anti-peptide sera contain antibodies directed against the CD4 attachment site on gp120 and interfere with this receptor-ligand interaction.
A high-performance liquid chromatographic (HPLC) method employing a C18 reversed-phase column, a mobile phase of sodium acetate and methanol, and an ultraviolet detector was developed for the analysis of 5-ethylpyrimidines and 5-methylpyrimidines in plasma. Samples were prepared for HPLC by sequential cation-exchange and anion-exchange column chromatography. Linear standard curves were obtained for samples containing 0.05-50 micrograms/ml 5-ethyl-2'-deoxyuridine and 5-ethyluracil, 0.05-10 micrograms ml 5-(1-hydroxyethyl)uracil, and 0.1-50 micrograms/ml thymidine, thymine and 5-hydroxymethyluracil. Applicability of the method to determination of the kinetics of 5-ethyl-2'-deoxyuridine elimination by the isolated perfused rat liver was demonstrated; clearance of the drug was 1.29 ml/min.
Brain incorporation of [1-14C]arachidonate ([14C]AA; 170 microCi/kg), [1-14C]docosahexaenoate ([14C]DA; 100 microCi/kg), or [9,10-3H]palmitate ([3H]PA; 6.4 mCi/kg) infused intravenously for 5 min was examined in the awake rat following systemic administration of the cholinomimetic arecoline (15 mg/kg i.p.). The rat was killed 15 min after infusion, and the brain was removed, frozen, and prepared for biochemical analysis and autoradiography. Brain radioactivity, normalized for plasma exposure, was increased by 41 and 45% in arecoline-treated rats given [14C]AA and [14C]DA, respectively. Pretreatment with atropine prevented the increase in fatty acid incorporation. Arecoline treatment had no effect on brain incorporation of [3H]PA. Quantitative autoradiography indicated regionally selective increases in brain [14C]AA and [14C]DA incorporation in response to arecoline. The results suggest that intravenously administered radiolabeled fatty acids can be used to study neurotransmitter-stimulated brain lipid metabolism in vivo.
Previous studies have indicated that the area postrema (AP) of the rat is necessary for the development of chronic angiotensin-dependent hypertension. The present study assesses the role of the AP in the maintenance of arterial pressure during hemorrhage. Sprague-Dawley rats were given sham or AP lesions 1 wk before the experiment. They were instrumented with femoral arterial and venous catheters 2 days before the experiment. On the day of the experiment, base-line mean arterial pressure (MAP) was measured for 1 h before hemorrhage. During the following 45 min, each rat was subjected to one 7-ml/kg hemorrhage every 15 min for a total of three hemorrhages. MAP was monitored by computerized data acquisition. As shown previously, MAP was slightly but significantly lower in AP-lesion rats compared with sham-lesion rats before the hemorrhage procedure. In AP-lesion rats, hemorrhage resulted in a significantly greater fall in arterial pressure than in sham-lesion rats. In spite of larger drops in pressure in AP-lesion rats, hemorrhage caused equivalent increases in plasma renin and vasopressin in both groups. In AP-lesion rats compared with sham-lesion rats, significant bradycardia was present before hemorrhage. Hemorrhage caused bradycardia in both sham- and AP-lesion rats relative to the prehemorrhage heart rates, but AP-lesion rats showed greater bradycardia than did sham-lesion rats during every time period. We conclude that the AP may play an important role in the defense of arterial pressure against hemorrhage.
The breakdown of 5-fluoro-2'-deoxyuridine (FdUrd) to 5-fluorouracil (FUra) is catalyzed by the pyrimidine nucleoside phosphorylases, uridine phosphorylase and thymidine phosphorylase. The effects of nucleoside phosphorylase inhibitors on FdUrd and FUra elimination by the isolated perfused rat liver were investigated. The inhibitor was injected into the perfusion reservoir 5 min before FdUrd or FUra, and serial perfusion fluid samples were collected for fluoropyrimidine analysis. The disappearance of each fluoropyrimidine followed Michaelis-Menten kinetics, as shown previously. 6-Benzyl-2-thiouracil, a thymidine phosphorylase-selective inhibitor, and 1-(2'-deoxy-beta-D-glucopyranosyl)thymine, a uridine phosphorylase-selective inhibitor, each decreased the rate of FdUrd disappearance (apparent Ki, 1.4-1.6 and 3.8 mM, respectively) but had no direct effect on FUra disappearance. However, 6-benzyl-2-thiouracil decreased the peak concentration of FUra derived from administered FdUrd and increased the t 1/2 of disappearance of derived FUra due to its delayed formation. 2,6-Dihydroxypyridine, a uridine phosphorylase-selective inhibitor, decreased the rate of FdUrd disappearance (apparent Ki, 12.4-16.2 microM) and directly inhibited FUra elimination (apparent Ki, 4.3-5.3 microM). 2,4-Dihydroxypyridine, which does not inhibit pyrimidine nucleoside phosphorylases, directly inhibited FUra elimination (apparent Ki, 77 microM) and also decreased the rate of FdUrd disappearance, possibly due to product (FUra) inhibition. It was concluded that the hepatic elimination of FdUrd is slowed by pyrimidine nucleoside phosphorylase inhibitors and that some of these drugs block FUra, as well as FdUrd, elimination.
The influence of dose and hepatic blood flow on the elimination of 5-fluoro-2'-deoxyuridine (FdUrd) by the isolated perfused rat liver were investigated. FdUrd (1-20 mg; 4-81 mumol) was injected into the perfusion reservoir and serial samples were collected for chromatographic determination of plasma FdUrd and 5-fluorouracil concentrations. The decrease in FdUrd concentration from values above 100 microM was linear with time (apparent zero order); at concentrations below 30-40 microM the decline became exponential (apparent first order). Semilogarithmic plots of FdUrd concentration/dose versus time obtained with different doses were not superposable, indicating Michaelis-Menten elimination. At a perfusion rate of 20 ml/min, the apparent Vmax and Km for FdUrd disappearance were 14-19 nmol/ml/min and 161-194 microM, respectively. FdUrd clearance during first-order elimination was 8-11 ml/min. After FdUrd administration, 5-fluorouracil concentration reached 10-15% of the initial FdUrd concentration, then decreased with a half-life of 4-7 min. Fifty-four % of the dose of [2-14C]FdUrd was converted to 14CO2. At a dose of 20 mg, first-order clearance of FdUrd increased from 7 to 12 ml/min as hepatic flow increased from 10 to 30 ml/min. Less than 1% of the dose of [6-3H]FdUrd was incorporated into macromolecules. It was concluded that hepatic elimination of FdUrd is dependent on both dose and blood flow.
Cerebral microvessels consisting predominantly of capillaries and small arterioles (less than 30 micron dia.) were isolated from the cerebral cortex and cerebellum of 3-month-old mice. Lipids were extracted from both microvascular and brain parenchymal fractions and the major phospholipid classes (choline phosphoglyceride, ethanolamine phosphoglyceride, inositol phosphoglyceride, serine phosphoglyceride, and sphingomyelin) separated by 2-dimensional TLC. Comparison of mol % determined by phosphate analysis of each phospholipid revealed significant differences in membrane composition of ethanolamine phosphoglyceride, inositol phosphoglyceride, and sphingomyelin between microvascular and parenchymal components of the central nervous system. Moreover, the choline phosphoglyceride/sphingomyelin mol ratio, one of three determinants of membrane fluidity, is significantly lower for microvessel membrane than for membranes of the brain parenchyma.
The kinetics of conversion of 5-fluoro-2'-deoxyuridine (FdUrd) to 5-fluorouracil (FUra) by isolated rat intestinal epithelial cells was investigated. Also, the effects of potential inhibitors of this reaction, which is catalyzed by uridine phosphorylase and thymidine phosphorylase, were determined. A 2.5% suspension of isolated cells was incubated with FdUrd or FUra, and at specific times cells were lysed with perchloric acid and fluoropyrimidines were determined by high-performance liquid chromatography. During a 25-min incubation with either FdUrd or FUra, the amount of drug in the incubation system (total volume 0.8 ml) fell by less than 5%. However, in the presence of FdUrd, the amount of FUra increased linearly over 25 min. The apparent Vmax and Km for FUra formation were 17-27 nmole/mg DNA/min and 1.6-2.5 mM, respectively. With each nucleoside phosphorylase inhibitor, the apparent Km increased but Vmax was unaffected. The apparent Ki values were as follows (in mM): 5-nitrouracil (an inhibitor of both uridine phosphorylase and thymidine phosphorylase), 0.12; 4-thiothymine (a uridine phosphorylase-selective inhibitor), 1.52; and 6-benzyl-2-thiouracil (a thymidine phosphorylase-selective inhibitor), 0.73. It was concluded that intestinal epithelial cells are capable of degrading FdUrd to FUra and that the cells possess both uridine phosphorylase and thymidine phosphorylase activity.
The influences of dose and hepatic blood flow on the elimination of 5-fluorouracil (FUra) by the isolated perfused rat liver were investigated. FUra was injected into the perfusion reservoir and then serial blood samples were collected over 2-3 h. FUra concentration was determined chromatographically. In some experiments, the conversion of [2-14C]FUra to 14CO2 was also determined. With livers perfused at 20 ml/min, the initial decrease in plasma FUra concentration was linear with time (apparent zero-order kinetics); at concentrations below about 25 microM, the decrease became exponential (apparent first-order kinetics). Semilogarithmic plots of FUra concentration/dose versus time obtained with different doses were not superposable, consistent with saturable (Michaelis-Menten) elimination. Vmax and Km were 6-11 nmol/ml/min and 33-45 microM, respectively. Hepatic clearance during first-order elimination was close to 20 ml/min. About 84% of the dose was converted to CO2, indicating that catabolic metabolism was the principal route of elimination. As hepatic blood flow increased from 10 to 30 ml/min, Vmax was unchanged but Km decreased progressively from 84 to 32 microM, and clearance increased from 12 to 29 ml/min. It was concluded that hepatic FUra elimination is highly dependent upon both dose and blood flow.
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The kinetics of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluorouracil (FUra) disposition after bolus intravenous injection were determined in anesthetized rhesus and cynomolgus monkeys. FdUrd disappearance from plasma was an apparent triexponential process with average half-lives of 0.5, 2, and 8 min; FUra disappearance was biphasic with average half-lives of 2 and 13 min. After FdUrd injection, FUra reached peak plasma concentrations of 15-30% of the initial FdUrd concentrations within 3 min, and then disappeared more slowly than FdUrd. Total FdUrd clearance fell from 105 to 73 to 56 ml/kg/min as the dose increased from 10 to 20 to 40 mg/kg. Metabolic clearance was about 85% of total clearance and fell similarly with increasing dosage. Total and metabolic FUra clearances were about 30% of FdUrd values at an equimolar dose. Renal FdUrd clearance exceeded glomerular filtration rate and was decreased by probenecid, indicating tubular secretion; renal FUra clearance was close to glomerular filtration rate. There was no apparent correlation between dose and renal clearance or volume of distribution. It was concluded that FdUrd, like FUra, is eliminated primarily by a dose-dependent process. The metabolic basis of the dose-dependent kinetics remains to be determined.
A gas-liquid chromatographic method employing on-column alkylation and a nitrogen-sensitive detector was developed for the analysis of 5-fluoro-2'-deoxyuridine, 5-fluorouridine, and 5-fluorouracil in plasma and urine. Samples (0.72 ml) containing the fluoropyrimidine and internal standard (5-chloro-2'-deoxyuridine for nucleoside analyses and 6-methyluracil for 5-fluorouracil analyses) were prepared for gas-liquid chromatography by sequential cation-exchange and anion-exchange column chromatography. Recoveries of fluoropyrimidines were 71-95% over the concentration ranges studied. The dried eluate from the anion-exchange column was dissolved in p-tolyltrimethylammonium hydroxide in methanol before gas-liquid chromatographic analysis. Columns packed with either 3% SP-2100 on Supelcoport or 3% OV-1 on Gas-Chrom Q were suitable for nucleoside analyses; a column packed with 0.75% Carbowax 20M-5% KOH on Chromsorb G was used for 5-fluorouracil analyses. The fluoropyrimidine nucleosides were well separated from each other and from the potentially interfering endogenous compounds 2'-deoxyuridine and uridine; 5-fluorouracil was well separated from uracil. Linear standard curves (peak area ratio method) were obtained for plasma containing 0.025 to 20 micrograms FdUrd (0.1 to 81 microM) or 0.05 to 1.0 microgram FUrd (0.2 to 3.8 microM), and for urine containing 0.2 to 1.0 microgram (0.8 to about 4 microM) of the nucleosides. Standard curves for 5-fluorouracil (1.5 to 7.9 microM) and 2'-deoxyuridine (0.9 to 4.4 microM) were also linear. A measurable amount of 5-fluorouracil, equivalent to 4 to 7% of the 5-fluoro-2'-deoxyuridine injected, was formed from the nucleoside on the gas-liquid chromatographic column, requiring correction of 5-fluorouracil concentrations measured in the presence of 5-fluoro-2'-deoxyuridine.
An easily constructed arterial catheter was designed for use in experiments requiring serial blood sampling from conscious, free-moving animals (Fischer 344 rat). It can be implanted chronically, at least several days before use, into the femoral artery, without interference of movement or damage by the animal. The catheter assembly is worn subdermally until the distal portion is exteriorized for blood sampling. The proximal tip of the catheter is occluded with a removable plug before implantation, thereby eliminating the necessity of daily flushing prior to use. The catheter has been used extensively in studies of the blood-brain barrier involving the bolus injection of [14C]sucrose into the jugular vein of conscious, unrestrained rats. Tracer concentration in plasma and whole blood was subsequently determined from arterial blood samples collected serially over a 20-min period following injection of the tracer.
While studying an experimental retinal immunopathy, we searched for a vascular tracer that would permit us to test the anatomical and functional status of retinal and choroidal vessels by ophthalmoscopy, light microscopy, and electron microscopy in the same eye. We found that colloidal carbon, administered intravenously, can be seen ophthalmoscopically in the choroidal and retinal circulations, and abnormal deposits of it can be visualized in vivo. The ophthalmoscopic findings can be corroborated and extended by stereomicroscopy, and later the same specimens can be used for light and transmission electron microscopical studies. This tracer, thus, would allow the correlation of the ophthalmoscopic appearance of a clinical lesion with its histological and ultrastructural substrata.
Significantly elevated levels of sodium fluorescein (MW 376) were found only in the brains of conscious rats made considerably hyperthermic (colonic temperatures greater than 41.0 degrees C) by exposure to ambient heat (42 +/- 2 degrees C) for 90 min or 2450 MHz CW microwave energy at 65 mW/cm2 (SAR approximately equal to 13.0 W/kg) for 30 or 90 min. For microwave-exposed rats, fluorescein levels within the cortex and hypothalamus appeared to increase with increasing duration of exposure. This trend was not apparent in the cerebellum or medulla. Exposure to ambient heat resulted in increased fluorescein with the cortex, hypothalamus and medulla, but not the cerebellum, and, in general, ambient heat was not as effective as microwave energy in raising tracer concentrations within the brain. By far the greatest elevation of fluorescein dye in the brain occurred in those animals whose blood-brain barrier had been opened osmotically by intracarotid injection of 10 M urea. It is suggested that increased levels of sodium fluorescein found in the brain tissue of ambient heat and microwave-exposed rats most likely represent technically derived artifact and not a breakdown of the blood-brain barrier.
Alteration of blood-brain barrier (BBB) permeability by 2450 MHz CW microwaves was assessed semi-quantitatively after intravenous injection of horseradish peroxidase (HRP) and exposure of conscious, unrestrained rats to incident power densities of 0, 20 or 65 mW/cm2 for 30, 90 or 180 min. Additional rats were exposed to ambient heat (42 +/- 2 degrees C) for 30 or 90 min. None of the brain regions studied, with the exception of the normally leaky pineal gland, showed extracellular HRP leakage attributable to microwave or thermally-induced breakdown of the blood-brain barrier. The mean ratio of HRP-labeled microvessel endothelium/total number of microvessels counted was determined for each brain region. Mean values for the cortex, hypothalamus, cerebellum and medulla of microwave-exposed and heated rats were consistently below those of corresponding sham levels. This decrease appeared to correlate inversely with power density and duration of exposure. Statistically significant deviation (P less than 0.05) from sham mean values occurred in the cortex, hypothalamus, cerebellum and medulla of animals made hyperthermic with ambient heat or exposure to microwaves at 65 mW/cm2 (specific absorption rate approximately equal to 13.0 W/kg) for 30 or 90 min. Additionally, electron microscopic evaluation of ultrathin sections taken from each of the 4 brain regions revealed no significant extravasation of HRP indicative of microwave or ambient heat-induced disruption of the blood-brain barrier.
Intravenously injected [14C]sucrose was used as a small molecular weight (342 daltons), hydrophilic tracer for determination of 2450 MHz CW microwave and ambient heat effects on rat blood-brain barrier permeability in the cerebral cortex, hypothalamus, cerebellum and medulla. The tracer was injected 4 min following exposure of conscious, unrestrained rats to microwaves at 0 or 65 mW/cm2 for 30 or 90 min (SAR approximately equal to 13.0 W/kg) or to ambient heat (42 +/- 2 degrees C) for 90 min. Comparison of mean permeability-surface area products (PA) and uptake ratios between sham and microwave-exposed animals revealed a statistically significant (P less than 0.05) decrease of both PA and uptake ratios for the hypothalamus, cerebellum and medulla of rats exposed to microwaves for 30 min. This decrease was not apparent for rats exposed to microwaves for 90 min. A pertinent observation, with regard to this latter group of animals, was the increased circulating levels of the tracer when colonic temperature was raised to approximately 41.4 degrees C or higher.